Microfluidic devices having a protein renaturation component and methods for using the same are provided. Aspects of the present disclosure include microfluidic devices that include a separation medium with a first flow path and a protein renaturation component in fluid communication with the separation medium and having a second flow path. Also provided are methods of using the devices as well as systems and kits that include the devices. The devices, systems and methods find use in a variety of different applications, including diagnostic and validation assays.
